The present invention relates to a gene responsible for onset of Parkinson""s disease. Since it was found that Parkinson""s disease patients have a deletion in part of the gene, the gene of this invention is significantly useful as a gene for diagnosing Parkinson""s disease, and a protein and a pharmaceutically active agent etc., obtainable from the inventive gene has usability in preventing and treating Parkinson""s disease.
Generally, it is often considered that one or more gene is responsible for various chronic progressive diseases. Isolating the gene or genes responsible for these diseases not only enables one to facilitate prenatal or postnatal diagnosis but also enables to perform gene therapy for the disease based on the remarkable progress and development of gene therapy as seen today.
Parkinson""s disease is a chronic disease. xcex1-synuclein reported in 1997 has so far been the only gene that has been found to be responsible for Parkinson""s disease. It is reported that some people having Italian ancestry suffer from autosomal dominant Parkinson""s disease due to mutation of this gene. There is, however, limitation in diagnosing Parkinson""s disease even with use of this gene. Therefore, what has been adopted at present as a diagnosis for Parkinson""s disease is merely a clinical approach based on neurodegenerative symptoms such as resting tremor, rigidity, akinesia, and disturbance of the righting ref lux, and a levodopa-responsive or dopaminergic compound (agonist) has been administered as a symptomatic treatment. So far no drastic therapy has been performed for treating Parkinson""s disease.
The present invention has been made in view of the above. An object of this invention is to provide an isolated DNA or gene or gene fragment that is responsible for Parkinson""s disease and is useful in diagnosing and treating the disease etc.; a recombinant vector; a protein or polypeptide; a monoclonal antibody or polyclonal antibody; a primer or probe or immobilized nucleic acid or DNA chip; and an oligonucleotide, or the like.
The isolated DNA or gene according to this invention that has overcome the above problems residing in the prior art is:
{circle around (1)} An isolated DNA or gene: comprising a full-length base sequence according to the SEQ ID. Nos. 1 or 3 (SEQ ID No. 3 does not include a base portion 636 to 719 (corresponding to exon 5 which is described later) of SEQ ID. No. 1, namely, a variant thereof according to alternative splicing), or a partial sequence thereof, or a base sequence hybridizable thereto or hybridizable with a complementary strand thereof, and being associated with Parkinson""s disease.
Further, the inventive DNA or gene may include a DNA or gene or gene fragment having the following features {circle around (2)} to {circle around (8)}, in addition to {circle around (1)}.
{circle around (2)} An isolated DNA or gene: comprising the base sequence of {circle around (1)}, or the full-length base sequence thereof, or the base sequence partially thereof, and the isolated DNA or gene whose gene defect is responsible for Parkinson""s disease, or comprising a base sequence hybridizable thereto or hybridizable with a complementary strand thereof.
An isolated DNA or gene comprising the base sequence of {circle around (1)} or {circle around (2)}, the isolated DNA or gene being variant thereof by alternative splicing, and being associated with Parkinson""s disease, or the isolated DNA or gene comprising a base sequence hybridizable thereto or hybridizable with a complementary strand thereof.
{circle around (4)} A gene comprising the base sequence of any one of {circle around (1)} to {circle around (3)} whose gene product encodes a protein having a substantially equivalent function to a protein comprising amino acids 1 to 465 of SEQ ID. No. 2 or a protein comprising amino acids 1 to 437 of SEQ ID. No. 4.
{circle around (5)} An isolated DNA or gene comprising a gene where an exonic deletion, a nonsense base substitute, a missense base substitute, a base deletion, a base addition, a base insertion, a splicing abnormality and/or a frameshift with respect to the base sequence has occurred in any one of {circle around (1)} to {circle around (4)}; or comprising a base sequence hybridizable thereto or hybridizable with a, complementary strand thereof, and the isolated DNA or gene being associated with Parkinson""s disease.
{circle around (6)} An isolated DNA or a gene, or a gene fragment comprising a partial base sequence of the DNA or the gene of any one of claims {circle around (1)} to {circle around (5)}, or an isolated DNA or a gene or a gene fragment comprising a base sequence hybridizable thereto or hybridizable with a complementary strand thereof.
{circle around (7)} A gene encoding a protein (a) or (b) comprising:
(a) the protein comprising amino acids 1 to 465 of SEQ ID. No. 1;
(b) the protein in which one or more amino acid(s) of the amino acid sequence is or are deleted, substituted, or added, and the protein being associated with Parkinson""s disease.
{circle around (8)} A gene encoding a protein (c) or (d):
(c) the protein comprising amino acids 1 to 437 of SEQ ID. No. 4;
(d) the protein in which one or more amino acid(s) of the amino acid sequence is or are deleted, substituted, or added, and the protein being associated with Parkinson""s disease.
The full-length base sequence SEQ ID. No. 1 is such that eleven introns are intervened among twelve exons on the genome; and encodes a protein having 1 to 465 amino acid sequence in a part (102 to 1496) of the base sequence. The base sequence of the intron in a boundary region between the exon and the intron has the following arrangement:
the intron intervening between exon 1 and exon 2 has a base sequence shown in SEQ ID. No. 9 adjacent to the 3xe2x80x2 end of the exon 1, and has a base sequence shown in SEQ ID. No. 10 adjacent to the 5xe2x80x2 end of the exon 2;
the intron intervening between exon 2 and exon 3 has a base sequence shown in SEQ ID. No. 11 adjacent to the 3xe2x80x2 end of the exon 2, and has a base sequence shown in SEQ ID. No. 12 adjacent to the 5xe2x80x2 end of the exon 3;
the intron intervening between exon 3 and exon 4 has a base sequence shown in SEQ ID. No. 13 adjacent to the 3xe2x80x2 end of the exon 3, and has a base sequence shown in SEQ ID. No. 14 adjacent to the 5xe2x80x2 end of the exon 4;
the intron intervening between exon 4 and exon 5 has a base sequence shown in SEQ ID. No. 15 adjacent to the 3xe2x80x2 end of the exon 4, and has a base sequence shown in SEQ ID. No. 16 adjacent to the 5xe2x80x2 end of the exon 5;
the intron intervening between exon 5 and exon 6 has a base sequence shown in SEQ ID. No. 17 adjacent to the 3xe2x80x2 end of the exon 5, and has a base sequence shown in SEQ ID. No. 18 adjacent to the 5xe2x80x2 end of the exon 6;
the intron intervening between exon 6 and exon 7 has a base sequence shown in SEQ ID. No. 19 adjacent to the 3xe2x80x2 end of the exon 6, and has a base sequence shown in SEQ ID. No. 20 adjacent to the 5xe2x80x2 end of the exon 7;
the intron intervening between exon 7 and exon 8 has a base sequence shown in SEQ ID. No. 21 adjacent to the 3xe2x80x2 end of the exon 7, and has a base sequence shown in SEQ ID. No. 22 adjacent to the 5xe2x80x2 end of the exon 8;
the intron intervening between exon 8 and exon 9 has a base sequence shown in SEQ ID. No. 23 adjacent to the 3xe2x80x2 end of the exon 8, and has a base sequence shown in SEQ ID. No. 24 adjacent to the 5xe2x80x2 end of the exon 9;
the intron intervening between exon 9 and exon 10 has a base sequence shown in SEQ ID. No. 25 adjacent to the 3xe2x80x2 end of the exon 9, and has a base sequence shown in SEQ ID. No. 26 adjacent to the 5xe2x80x2 end of the exon 10;
the intron intervening between exon 10 and exon 11 has a base sequence shown in SEQ ID. No. 27 adjacent to the 3xe2x80x2 end of the exon 10, and has a base sequence shown in SEQ ID. No. 28 adjacent to the 5xe2x80x2 end of the exon 11; and
the intron intervening between exon 11 and exon 12 has a base sequence shown in SEQ ID. No. 29 adjacent to the 3xe2x80x2 end of the exon 11, and has a base sequence shown in SEQ ID. No. 30 adjacent to the 5xe2x80x2 end of the exon 12.
In addition, a recombinant vector comprising the DNA fragment or the gene of any one of {circle around (1)} to {circle around (8)} may be included in the scope of this invention.
The protein which has overcome the above problem is (i) a protein comprising amino acids 1 to 465of SEQ ID. No. 1; or (ii) a protein comprising 1 to 437 amino acid sequence in the sequence ID. No. 2, the protein being associated with Parkinson""s disease; or a protein having a substantially equivalent function thereto.
More specifically, the protein or polypeptide according to this invention may embrace the following aspects (ii) to (viii).
(ii) A protein expressed by the gene of any one of {circle around (1)} to {circle around (4)}, the protein being associated with Parkinson""s disease, or having an identical function thereto or a substantially equivalent function thereto.
(iii) A protein comprising an amino acid sequence translated by the gene of {circle around (5)}, the protein being associated with Parkinson""s disease, or having an identical function thereto or a substantially equivalent function thereto.
(iv) A protein comprising the amino acid sequence of (iii) in which an amino acid is substituted, deleted, or added at least at one position, and the protein being associated with Parkinson""s disease.
(v) A protein comprising the amino acid sequence of any one of (ii) to (iv) comprising: a ubiquitin-like 1 to 72 amino acid sequence partially included in SEQ ID. No. 1; and a zinc-finger-protein-like 418 to 449 amino acid sequence partially included in SEQ ID. No. 1.
(vi) A protein (a) or (b):
(a) the protein comprising amino acids 1 to 465 of SEQ ID. No. 1;
(b) the protein in which one or more amino acid(s) of the amino acid sequence is or are deleted, substituted, or added, and the protein being associated with Parkinson""s disease.
(vii) A protein (c) or (d):
(c) the protein comprising 1 to 437 ID. No. 2;
(d) the protein in which one or more amino acid(s) of the amino acid sequence is or are deleted, substituted, or added, and the protein being associated with Parkinson""s disease.
(viii) A polypeptide or a protein consisting of a partial fragment of the amino acid sequence of any one of (i) (vii), or comprising the partial fragment thereof, or the full-length amino acid sequence thereof.
In addition, a monoclonal antibody or a polyclonal antibody against the protein of any one of (i) to (viii) may be included in the scope of this invention.
Further, a primer, or a probe, or an immobilized nucleic acid, or a DNA chip according to this invention may preferably be used for the following purposes (I) to (IV):
(I) for use in detecting a base sequence, a genetic mutation, a deletion, and/or an expression amount of the DNA or the gene of any one of {circle around (1)} to {circle around (8)}, or for use in concentration thereof;
(II) for use in detecting a base sequence, a genetic mutation, a deletion, and/or an expression amount of RNA which is subjected to transcription and subjected to processing from the DNA or the gene of any one of {circle around (1)} to {circle around (8)}, or for use in concentration thereof;
(III) for use in detecting a base sequence, a genetic mutation, and/or a deletion of the exon of SEQ ID. No. 1 or 3, or for use in haplotyping a locus thereof; or
(IV) for use in detecting a base sequence, a genetic mutation, and/or a deletion of the aforemention intron, or for use in haplotyping a locus thereof.
Specifically, at least one of the fourteen sets of primers or probes shown in the following (1) to (14) can be used.
(1) A primer or a probe for use in detecting a base sequence of the intron adjacent to the exon 1 of the gene being associated with Parkinson""s disease of {circle around (1)}, or a locus thereof, the primer or probe comprising the following base sequence:
a base sequence of SEQ ID. No. 31 in the 5xe2x80x2-3xe2x80x2 direction of SEQ ID. No. 1 on the genome, and
a base sequence of SEQ ID. No. 32 in the 5xe2x80x2-3xe2x80x2 direction on a complementary strand of SEQ ID. No. 1 on the genome.
(2) A primer or a probe for use in detecting a base sequence of an intron adjacent to the exon 2 of the gene being associated with Parkinson""s disease of {circle around (1)}, or a locus thereof, the primer or the probe comprising the following base sequence:
a base sequence of SEQ ID. No. 33 in the 5xe2x80x2-3xe2x80x2 direction of SEQ ID. No. 1 on the genome, and
a base sequence of SEQ ID. No. 34 in the 5xe2x80x2-3xe2x80x2 direction on a complementary strand of SEQ ID. No. 1 on the genome.
(3) A primer or a probe f or use in detecting a base sequence of an intron adjacent to the exon 3 of the gene being associated with Parkinson""s disease of {circle around (1)}, or a locus thereof, the primer or the probe comprising the following base sequence:
a base sequence of SEQ ID. No. 35 in the 5xe2x80x2-3xe2x80x2 direction of SEQ ID. No. 1 on the genome, and
a base sequence of SEQ ID. No. 36 in the 5xe2x80x2-3xe2x80x2 direction on a complementary strand of SEQ ID. No. 1 on the genome.
(4) A primer or a probe for use in detecting a base sequence of the intron adjacent to the exon 4 of the gene being associated with Parkinson""s disease of {circle around (1)}, or a locus thereof, the primer or probe comprising the following base sequence:
a base sequence of SEQ ID. No. 37 in the 5xe2x80x2-3xe2x80x2 direction of SEQ ID. No. 1 on the genome, and
a base sequence of SEQ ID. No. 38 in the 5xe2x80x2-3xe2x80x2 direction on a complementary strand of SEQ ID. No. 1 on the genome.
(5) A primer or a probe for use in detecting a base sequence of an intron adjacent to the exon 4 of the gene being associated with Parkinson""s disease of {circle around (1)}, or a locus thereof, the primer or the probe comprising the following base sequence:
a base sequence of SEQ ID. No. 39 in the 5xe2x80x2-3xe2x80x2 direction of SEQ ID. No. 1 on the genome, and
a base sequence of SEQ ID.No. 40 in the 5xe2x80x2-3xe2x80x2 direction on a complementary strand of SEQ ID. No. 1 on the genome.
(6) A primer or a probe for use in detecting a base sequence of an intron adjacent to the exon 5 of the gene being associated with Parkinson""s disease of {circle around (1)}, or a locus thereof, the primer or the probe comprising the following base sequence:
a base sequence of SEQ ID. No. 41 in the 5xe2x80x2-3xe2x80x2 direction of SEQ ID. No. 1 on the genome, and
a base sequence of SEQ ID. No. 42 in the 5xe2x80x2-3xe2x80x2 direction on a complementary strand of SEQ ID. No. 1 on the genome.
(7) A primer or a probe for use in detecting a base sequence of the intron adjacent to the exon 6 of the gene being associated with Parkinson""s disease of {circle around (1)}, or a locus thereof, the primer or probe comprising the following base sequence:
a base sequence of SEQ ID. No. 43 in the 5xe2x80x2-3xe2x80x2 direction of SEQ ID. No. 1 on the genome, and
a base sequence of SEQ ID. No. 44 in the 5xe2x80x2-3xe2x80x2 direction on a complementary strand of SEQ ID. No. 1 on the genome.
(8) A primer or a probe for use in detecting a base sequence of an intron adjacent to the exon 7 of the gene being associated with Parkinson""s disease of {circle around (1)}, or a locus thereof, the primer or the probe comprising the following base sequence:
a base sequence of SEQ ID. No. 45 in the 5xe2x80x2-3xe2x80x2 direction of SEQ ID. No. 1 on the genome, and
a base sequence of SEQ ID. No. 46 in the 5xe2x80x2-3xe2x80x2 direction on a complementary strand of SEQ ID. No. 1 on the genome.
(9) A primer or a probe for use in detecting a base sequence of an intron adjacent to the exon 7 of the gene being associated with Parkinson""s disease of {circle around (1)}, or a locus thereof, the primer or the probe comprising the following base sequence:
a base sequence of SEQ ID. No.47 in the 5xe2x80x2-3xe2x80x2 direction of SEQ ID. No. 1 on the genome, and
a base sequence of SEQ ID. No. 48 in the 5xe2x80x2-3xe2x80x2 direction on a complementary strand of SEQ ID. No. 1 on the genome.
(10) A primer or a probe for use in detecting a base sequence of the intron adjacent to the exon 8 of the gene being associated with Parkinson""s disease of {circle around (1)}, or a locus thereof, the primer or probe comprising the following base sequence:
a base sequence of SEQ ID. No. 49 in the 5xe2x80x2-3xe2x80x2 direction of SEQ ID. No. 1 on the genome, and
a base sequence of SEQ ID. No. 50 in the 5xe2x80x2-3xe2x80x2 direction on a complementary strand of SEQ ID. No. 1 on the genome.
(11) A primer or a probe for use in detecting a base sequence of an intron adjacent to the exon 9 of the gene being associated with Parkinson""s disease of {circle around (1)}, or a locus thereof, the primer or the probe comprising the following base sequence:
a base sequence of SEQ ID. No. 51 in the 5xe2x80x2-3xe2x80x2 direction of SEQ ID. No. 1 on the genome, and
a base sequence of the SEQ ID. No. 52 in the 5xe2x80x2-3xe2x80x2 direction on a complementary strand of SEQ ID. No. 1 on the genome.
(12) A primer or a probe for use in detecting a base sequence of an intron adjacent to the exon 10 of the gene being associated with Parkinson""s disease of {circle around (1)}, or a locus thereof the primer or the probe comprising the following base sequence:
a base sequence of SEQ ID. No. 53 in the 5xe2x80x2-3xe2x80x2 direction of SEQ ID. No. 1 on the genome, and
a base sequence of SEQ ID. No. 54 in the 5xe2x80x2-3xe2x80x2 direction on a complementary strand of SEQ ID. No. 1 on the genome.
(13) A primer or a probe for use in detecting a base sequence of the intron adjacent to the exon 11 of the gene being associated with Parkinson""s disease of {circle around (1)}, or a locus thereof, the primer or probe comprising the following base sequence:
a base sequence of SEQ ID. No. 55 in the 5xe2x80x2-3xe2x80x2 direction of SEQ ID. No. 1 on the genome, and
a base sequence of SEQ ID. No. 56 in the 5xe2x80x2-3xe2x80x2 direction on a complementary strand of SEQ ID. No. 1 on the genome.
(14) A primer or a probe for use in detecting a base sequence of an intron adjacent to the exon 12 of the gene being associated with Parkinson""s disease of {circle around (1)}, or a locus thereof, the primer or the probe comprising the following base sequence:
a base sequence of SEQ ID. No. 57 in the 5xe2x80x2-3xe2x80x2 direction of SEQ ID. No. 1 on the genome, and
a base sequence of SEQ ID. No. 58 in the 5xe2x80x2-3xe2x80x2 direction on a complementary strand of SEQ ID. No. 1 on the genome.
Further, the present invention may include the following oligonucleotide, or an oligonucleotide analog, or a modified product thereof as shown in (a) to (c):
(a) the one which comprises a partial sequence of the base sequence of any one of {circle around (1)} to {circle around (8)}, or which is hybridizable with the base sequence of any one of {circle around (1)} to {circle around (8)}.
(b) the one for use in amplifying the full-length base sequence or the partial base sequence of any one of {circle around (1)} to {circle around (8)}, or the oligonucleotide for use in amplifying partially the full-length base sequence or the partial base sequence of any one of {circle around (1)} to {circle around (8)}, according to PCR method using a human RNA as a template, PCR method or RT-PCR method using a human cDNA as a template.
(c) the oligonucleotide for use in amplifying the base sequence comprising the exon in SEQ ID. No. 1 or No. 3 and the aforementioned intron which is adjacent to the exon according to PCR method, or the oligonucleotide for use in amplifying a part of the base sequence according to PCR method.